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What are the criteria for choosing PCR amplicon length?

Ideal amplicon length depends on many variables and design preferences. For standard PCR scientists generally design amplicons to be between 200–1000 bp. For quantitative PCR, standard amplicons range from 75–150 bp.  It is unlikely that an amplicon will be too short. However amplicons of >1000 bp may need extra time to be completed during the extension step.  The polymerization rate of the enzyme used in the reaction needs to be sufficient to copy the amplicon in its entirety, so for long amplicons, you may need to increase the extension cycle time.  The general rule of thumb is 1 minute/kb of amplicon. You will also need to optimize reaction component concentrations based on the length of the amplicon.
Tags:
  • Length
  • extension
  • amplicon size
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